Pathogens caught in the act - Dynamic processes in rapidly frozen cells studied in electron microscope

June 18, 2007

For the first time, cell infections can be studied “live“ by the use of cryo-electron tomography. Researchers at the Max Planck Institute of Biochemistry apply this technique to attain true-to-life three-dimensional images of a pox-virus infection and publish studies detailing the structure and motion of the malaria-causing Plasmodium in unrivaled detail. Both studies show that this new method can be used for medical purposes (’’Journal of Experimental Medicine’’ June 2007, ’’Public Library of Science One,’’ May 2007).

Viruses are incapable of reproduction on their own. They must infect cells of higher organisms to survive. The host cells are reprogrammed by the viral genome to multiply the viruses, which are then released to seek new cells to infect. In most cases, viral infections are very dangerous to humans and sometimes cause fatal diseases such as AIDS, smallpox, and measles. Scientists at the Max Planck Institute of Biochemistry, in co-operation with colleagues at the EMBL in Heidelberg, have been studying the process of vaccina virus infection of cultured fibroblast cells at a molecular level. Vaccina virus, used as a vaccine against smallpox, serves the scientists as a model organism on which they can apply the newly invented cryo-electron tomography technology to demonstrate the dynamic processes occurring in its cells.

Cryo-electron tomography is a special form of transmission electron microscopy (TEM) in which the samples are rapidly frozen and then placed in the electro microscope’s vacuum and examined at a resolution of up to 4 nanometers (1 nanometer is one millionth of a millimeter) in three dimensions. Marek Cyrklaff and his colleagues in the Department of Molecular Structural Biology have elaborated a time-resolved approach to study the biological processes in the cryo-electron microscope. Reconstruction of 3D images is combined with cutting-edge EM-technology masterminded in the department to map the structure of the cells. Three-dimensional macromolecular cell-structure models are then produced out of countless 2D EM images by the skilled scientists.

The vaccina virus cryo-tomograms show in unmatched detail what structural changes the virus undergoes in contact with the host cell before emitting its genome: after attachment to the outer cell membrane is achieved, the membrane layers loosen and the so-called lateral bodies break free of the virus core. Simultaneously, the dumbbell-shaped core itself changes form and becomes oval. The outer virus membrane spontaneously dissolves and the spikes, fastened hexagonally on the virus core, disperse themselves in a random formation on its surface. The DNA strands, previously condensed at inner aspect of the viral core, break free and now occupy the entirety of the core. The virus core penetrates the plasma membrane of the host cell, causing the side to burst. Now viral DNA is completely released into the host cell and sets about the task of reprogramming the cell signal system to reproduce more viruses.

In a second study the cooperation between the Martinsried scientists and researchers from the Hygiene Institute of the University of Heidelberg resulted in the discovery of until know unknown structural details in other human disease carriers. Malaria is the most common tropical disease; about 1 million humans die each year of malaria caused predominantly by Plasmodium falciparum. A severe problem in the countries threatened by malaria is that the germs develop immunity against medicines over time. Marek Cyrklaff and his colleagues under the guidance of Friedrich Frischknecht, are now recording up-to-date details about the structure of malaria-causing parasites. Plasmodium possesses differing sexual and asexual growth periods, which run varying reproduction cycles in different hosts (such as mosquitoes, humans and other mammals).

During the infection of a human by a mosquito bite so-called sporozoites are released through the mosquito saliva into the human skin and from there on into the blood flow. The unicellular sporozoites travel through the blood flow to the liver where they reproduce as merozoites, which beset the red blood cells and split there. In this process, hemoglobin is degraded and fever is caused. Sporozoites are lengthwise-stretched single-celled organisms, which can easily travel about the skin due to their snakelike movement. Additionally the parasite possesses a cell skeleton, which is created by, amongst others, microtubules, which are long, lengthwisestretched, parallel protein structures. The researchers were then able to show how the microtubules interact sideways with a ring at one of the poles. Through the use of the enormously magnified images, the scientists proved how the microtubules possess unusually thick walls, an unknown fact until then. The biologists then concluded that there must be an unknown protein, which gives the microtubules an unusually powerful flexibility.

This helps it bend shape and eases its passage when intercepting the host cell. The cellular model elaborated by the scientists for this study enables investigations with electron microscope of the entire cell in 2 and 3 dimensions without a need of cell sectioning. It therefore offers unusual prospects for studying intact cellular structures and processes in situ, of every part of a cell.

Both studies were made possible through the cooperation between structural biologists, virologists, parasitologists and bioinformaticians. They show how cryo-electron tomography is a useful and important tool in the examination of dynamic cellular processes, and in the creation of new, alternative medicine. [SPM/EMD]

Original Publications:

Marek Cyrklaff, Alexandros Linaroudis, Marius Boicu, Petr Chlanda , Wolfgang Baumeister, Gareth Griffiths, Jacomine Krijnse-Locker (2007). Whole Cell Cryo-Electron Tomography Reveals Distinct Disassembly Intermediates of Vaccinia Virus. Open Access Publication PLoSONE2(5):e420.doi:10.1371/journal.pone.0000420

Marek Cyrklaff, Mikhail Kudryashev, Andrew Leis, Kevin Leonard, Wolfgang Baumeister, Robert Menard, Markus Meissner, and Friedrich Frischknecht (2007). Cryoelectron tomography reveals periodic material at the inner side of subpellicular microtubules in apicomplexan parasites. Published Online 11 June 2007: DOI:10.1084/jem.20062405.


Marek Cyrklaff, PhD

Research Department Molecular Structural Biology

Eva-Maria Diehl, Public Relations

Tel: 089 8578 2824

Max Planck Institute of Biochemistry

Am Klopferspitz 18

82152 Martinsried

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